Dandidiasis is a widespread fungal infection caused by different Candida species especially in patients infected with human immunodeficiency virus (HIV), immunocompromised individuals, intra-venous drug abusers, patients undergoing hematopoietic stem cell and solid organ transplant recipients, and those who present underlying valvular heart diseases or prolonged use of intravenous catheters . Although Candida albicans is responsible for up to 78 % of nosocomial candidiasis , reports describing cases of Candida infections by non-albicans Candida species have been also documented . Consequently, it is important to identify the causative organism to the species level correctly. Rapid identification of Candida species in clinical laboratory is becoming increasingly important since the identification and discrimination of etho-logical agents for early treatment, and preventing the invasion is highly recommended . Due to the high degree of phenotypic similarity between Candida species, identification problems are imminent. Conventional approaches for identi-fication down to the species level are not very effective in detecting yeast . Therefore, Alternative molecular assay with high specificity, reproducibility and sensitivity are necessary [6, 7]. The objective of current study was to identify the common Candida species isolated from clinical samples by using both polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and amplification of hwp1 gene.
Materials and Methods
Clinical samples comprising of vaginal specimens, cutaneous, sputum, bronchoalveolar lavage (BAL), and blood cultures were recovered from suspected patients during routine clinical administration at several hospitals of Tehran and Sari, Iran. Initially, obtained specimens were inoculated onto Sabouraud dextrose agar (SDA, Difco) supplemented with chloramphenicol and incubated at 35°C for up to 2 days. The obtained strains were preliminarily identified to the species level based on phenotypic characterizations, i.e., colony color on CHROM agar Candida medium (CHROMagar Company, Paris, France), germ-tube formation, microscopic morphology on corn-meal agar (Difco laboratories, Detroit, Mich., USA) with 1 % tween 80. Identified strains were deposited to the reference culture collection of Invasive Fungi Research Center (IFRC), Sari, Iran.
Genomic DNA was extracted, using glass bead disruption. Briefly, 300 µl of lyses buffer (10 mM Tris, 1 mM EDTA (pH= 8), 1 % SDS, 100 mM NaCl, 2 % triton X-100), 300 µl of phenol chloroform (1:1) solution and equal to 300 µl of 0.5 mm diameter glass beads, were added to yeast. After 5 min of vigorous shaking which followed by 5 min centrifugation at 10000 rpm, the supernatant was isolated and transferred to a new tube and equal volume of chloroform was added, mixed gently, centrifuged and its supernatant was transferred to a new tube. For alcohol precipitation, 0.1 mL volume sodium acetate (pH= 5.2) and 2.5 mL volume of cold absolute ethanol were added and the mixture was gently shaken and centrifuged at 10000 rpm for 10 min at 4°C. After washing with 70 % ethanol, the pellets re-suspended in 100 µl TE buffer (10 mM Tris, 1 mM EDTA) and were stored at -20°C prior to use.
RCR- RFLP analysis
The PCR-RFLP method was performed as previously described . Briefly, PCR amplification of ITS1-5.8S-ITS2 rDNA regions was achieved using the universal primers ITS1 (5ʹ -TCC GTA GGT GAA CCT GCG G-3ʹ) and ITS4 (5ʹ -TCCTCCGCTTATTGATAT GC-3ʹ) (MWG-Biotech AG, Germany). PCR amplification was performed in a final volume of 50 µl. Each reaction consists of 2 µl template DNA, 0.5 µl of each primer at 25 pmol, 1.25 µl of dNTP (BIORON GmbH, Germany) at 5 mM, 0.5U Taq DNA polymerase (Roche Diagnostics GmbH, Mannheim, Germany) and 5 µl 10× PCR buffer. The PCR conditions were as follow: 35cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 1 min. Subsequently, PCR products were digested in a final reaction volume of 15μl containing 3μl water, 1.5μl buffer, 1U of restriction enzyme MspI (Fermentas, USA) and 10μl PCR product at 37˚C for 2h. Amplified and digested products were visualized by 1.5 % and 2 % agarose gel electrophoresis in TBE buffer (0.09 M Tris, 0.09 M boric acid and 2 mM EDTA, pH 8.3) respectively, and stained with ethidium bromide (0.5 µg/ml) and photographed.
Partial amplification of the hwp1 gene
Definitive species identification and discri-mination of all members of the C. albicans species complex (C. albicans, C. dubliniensis, C. africana, and C. stellatoidea) was performed by the amplification of the hyphal wall protein 1 (hwp1) gene as described by Romeo and Criseo . PCR amplification of hwp1 gene was achieved using the forward, 5′-GCTACCACTTCAGAATCATCATC-3′ and reverse, 5′-GCACCTTCAGTCGTAGAGACG-3′ primer pairs. The partial amplification of the hwp1 gene was used to distinguish C. albicans isolates (941 bp) from atypical form i.e., C. africana (750 bp) and C. dubliniensis (569bp). However, results were confirmed by sequencing of the ITS rDNA regions using previously described primers . Briefly, sequencing was performed on an ABI 3730xl automatic sequencer (Applied Biosystems, Foster City, CA) and adjusted using the SeqMan of Lasergene software (DNAStar Inc., USA.) and compared with GenBank database (http://www.ncbi.nlm.nih.gov/).
One hundred and fifty clinical specimens were collected from patients with candidiasis. Clinical samples were obtained from vagina (67.5 %), skin scraping (13.2 %), sputum (8.3 %), BAL (6.8 %), and ear discharge (4.2 %). Figure 1 demonstrates the patterns of ITS-RFLP for Candida strains after digestion with Msp I. The most prevalent species isolated from patients were C. albicans (n=67; 44.6 %), C. glabrata (n=30; 20 %), C. tropicalis (n= 20; 13.3 %), C. krusei (n=12; 8 %), C. parapsilosis (n=11; 7.3 %). PCR amplification of hwp1 gene was used for discrimination of C. albicans, C. dubliniensis, C. africana, and C. stellatoidea. Figure 2 shows the patterns of PCR amplification of hwp1 gene. Out of 67 C. albicans species, 6, 4 and were identical as C. dubliniensis and C. africana respe-ctively. There were no significant differences observed between the distribution and origin of Candida isolates. However, C. africana and C. glabrata were identified most in vaginal samples (P≤0.01).
Recently, the increased incidence of severe candidiasis has been attributed to the enlargement of the population of high risk patients, which is resulted from advances achieved by modern medicine, immunosuppressives, and broad-spectrum antibacterial drugs . Despite the advances in medical interventions and the development of new antifungal agents, the mortality rate remains high . Because of high degree of phenotypic similarity among species of Candida, identification problems are imminent . Identification down to the species level based on conventional criteria is time consuming and need expertise. This report provides the species distribution of clinical isolates of Candida species with molecular identification. In this study, C. albicans was the most common species associated with Candida infection (44.6 %), followed by C. glabrata (20 %) which is in agreement with other studies [2, 12, 13], however the overall percentage of non-albicans species (55.4 %) was higher than in previous reports . Shokohi et al. and Papon et al. Mentioned C. glabrata as the most common non-C. albicans species in their investigation [14, 15]. However, Alborzi et al. showed that he most abundant species isolated from Iranian patients were as follow;C. albicans (50 %), C. glabrata (21.4 %), C. dubliniensis (13.3 %), C. krusei (9.8%), C. kefyr (3.1 %), C. parapsilosis (1.6 %), and C. tropicalis (0.8 %) . These differences in rates might be attributable to differences in the representation of the populations studied and ecological and climatological factors. The present study found that Candida species isolated from patients identified as C. dubliniensis (n=6) and C. africana (n=4). Solimani et al, also showed that 7.1 % of species from C. albicans identified as C. dubliniensis, however C. africana strains were not found . In addition, Yazdanparast et al. believe that based on molecular tools, C. africana constituted 4.38 % of the vaginal isolates of the C. albicans species complex . In this study, we performed PCR-RFLP assay and amplification of hwp1 gene to identify the clinically important Candida species and we found that C. albicans as predominant species isolated from this clinical samples [6, 17, 18].
High frequency of non albicans Candida species and differences in levels of susceptibility to the antifungal agents are important issues in medicine. Therefore, to manage the Candida-related infections properly, molecular diagnostic methods would be fast, reliable and even cost-effective approaches for identification of Candida species.
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